Cerebroside sulfatase activator deficiency induced metachromatic leukodystrophy

Two siblings of consanguineous parents had presented with a variety of findings indicative of juvenile metachromatic leukodystrophy (MLD). However, instead of the expected profound deficiency of arylsulfatase A (ARS A), their enzyme levels were about half-normal, and enzyme from fibroblasts had properties identical with the properties of enzyme from normal fibroblasts. Nevertheless, the hydrolysis of cerebroside sulfate by growing fibroblasts was markedly attenuated. Supplementation of the fibroblasts with cerebroside sulfatase activator normalized the response in the loading test. These results imply that the fibroblasts, and by extension the patients, are deficient in activator. Although the defective catabolism of cerebroside sulfate and the clinical manifestations in these patients mimic MLD, the molecular basis is distinct from the classical forms of the disorder.     

Two cage-type lupin alkaloids from Sophora franchetiana

Two new cage-type lupin alkaloids, (?)-tsukushinamine-B and tsukushinamine-C, have been isolated from the fresh epigeal parts of Sophora franchetiana, along with (?)-cytisine, (?)-N-formylcytisine, (?)-rhombifoline, (?)-anagyrine, (?)-baptifoline and (±)-ammodendrine, as well as (?)-tsukushinamine-A. The structures of these novel tsukushinamine-type lupin alkaloids were determined by spectroscopic data and partly by a chemical reaction. Variations of the alkaloid contents in the seeds, seedlings and various parts of S. franchetiana were also examined.     

Proton nuclear-magnetic-resonance and resonance Raman studies of thermophilic cytochrome c-552 from Thermus thermophilus HB8

The pH and temperature dependences of the 270-MHz proton nuclear magnetic resonance and resonance Raman spectra of Thermus thermophilus cytochrome c-552 were studied. Observation of the NMR methyl signal of the iron-bound methionine indicates that a methionine residue is the sixth ligand of heme iron in both ferric and ferrous states, although the environment of this methionine is not similar to that in mitochondrial cytochrome c. The NMR methyl signal of the coordinated methionine in the ferrous state was observed even at 87 degrees C, indicating the retention of the methionine ligand at the sixth coordination position. None of resonance Raman lines in ferrous cytochrome c-552 at higher temperatures showed a prominant temperature-dependent frequency shift, which implies that the heme iron was still bound with strong ligands and retained the low-spin state. In either redox state overall thermal denaturation did not occur even at 87 degrees C, although the ferric form existed in thermal spin mixture of the low-spin and high-spin species at higher temperatures. The hyperfine-shifted NMR resonances of the ferric form indicated rapid exchange of the sixth ligand at alkaline pH in the process of a single-step alkaline isomerization.     

Alkaline isomerization of thermoresistant cytochrome c-552 and horse heart cytochrome c studied by absorption and resonance Raman spectroscopy.

The structure of the thermoresistant cytochrome c (552, Thermus thermophilus) has been investigated at neutral and alkaline pH by absorption and resonance Raman spectroscopy and compared with that of horse heart cytochrome c. The ligands of the ferricytochrome c-552 at neutral pH are considered to be histidine and methionine, whereas the ligands of ferrocytochrome c-552 are histidine and another nitrogen base, histidine or lysine. Ferric cytochrome c-552 undergoes an alkaline isomerization with a pK of 12.3 (25 degrees C), accompanied by a ligand exchange. Horse heart cytochrome c has at least three isomerization states at alkaline pH (pK 9.3, 12.9 and greater than 13.5 at 25 degrees C). The replacement of the sixth ligand may not be involved in the second isomerization. The thermodynamic parameters for the isomerization were also estimated. The entropy change upon isomerization of cytochrome c-552 is negative, whereas for that of horse heart cytochrome c the entropy change is positive.     

Kinetic study of isomerization of ferricytochrome c at alkaline pH

Kinetic studies of the isomerization reaction of horse heart ferricytochrome c between pH 8.5 and pH 12.1 have been carried out by using stopped-flow and rapid scanning stopped-flow techniques. Below pH 10, our results were in good agreement with the scheme proposed earlier (Davis, L. A., Schejter, A. and Hess, G. P. (1974) J. Biol. Chem. 249, 2624–2632). Above pH 10, another faster first-order process was observed, which suggested the existence of a transient species in the isomerization reaction between the species with and without a 695 nm band. The probable scheme of the isomerization reaction is considered to be

where H denotes a proton, the colored forms are the species predominant at neutral pH with a 695 nm band and the noncolored forms are the species without a 695 nm band. The transient species has a small 695 nm absorbance which suggests that the sixth ligand is still Met-80, although the protein conformation might be different from that at neutral pH.     

The distribution of 14c from [U-14c]glucose in mice using whole-body autoradiography.

Tissue distribution of radioactive carbon from [U-14C]glucose in the mouse in vivo was studied by whole-body autoradiography. The mice were frozen with Dry-Ice-acetone at 0.5, 1, 5 and 30 min, 1 and 24 hr and 1 and 3 weeks after intraperitoneal injection of [U-14C]glucose. Whole-sagittal sections of the frozen mouse, obtained by using a microtome in a cryostat, were dried in a cryostat and autoradiographed. The resulting dry autoradiographs are called untreated autoradiographs in the present work. The sections were then fixed in cold 6% (w/v) HClO4, dried at room temperature and again autoradiographed. Autoradiographs that have undergone this process are referred to as treated autoradiographs. In both untreated and treated autoradiographs, within 1 min following injection of the labeled glucose, the abdominal cavity had the highest autoradiographic density. At 1 hr, density became highest in Harder's, sublingual and duodenal glands, large intestinal mucosa and tongue, and after 3 weeks, no autoradiographic denisty was present.     

Fbxo45 Forms a Novel Ubiquitin Ligase Complex and Is Required for Neuronal Development

Fbxo45 is an F-box protein that is restricted to the nervous system. Unlike other F-box proteins, Fbxo45 was found not to form an SCF complex as a result of an amino acid substitution in the consensus sequence for Cul1 binding. Proteomics analysis revealed that Fbxo45 specifically associates with PAM (protein associated with Myc), a RING finger-type ubiquitin ligase. Mice deficient in Fbxo45 were generated and found to die soon after birth as a result of respiratory distress. Fbxo45^−/− embryos show abnormal innervation of the diaphragm, impaired synapse formation at neuromuscular junctions, and aberrant development of axon fiber tracts in the brain. Similar defects are also observed in mice lacking Phr1 (mouse ortholog of PAM), suggesting that Fbxo45 and Phr1 function in the same pathway. In addition, neuronal migration was impaired in Fbxo45^−/− mice. These results suggest that Fbxo45 forms a novel Fbxo45-PAM ubiquitin ligase complex that plays an important role in neural development.Ubiquitin-dependent proteolysis is indispensable for various biological processes (3, 40). Protein ubiquitylation is mediated by several enzymes that act in concert, with a ubiquitin ligase (E3) playing a key role in substrate recognition (14). E3 enzymes contain specific structural motifs that mediate recruitment of a ubiquitin-conjugating enzyme (E2), with these motifs including HECT, RING finger, U-box, and PHD finger domains (30). The SCF complex consists of Skp1 (adaptor subunit), Cul1 (scaffold subunit), an F-box protein (substrate recognition subunit), and Rbx1 (also known as Roc1 or Hrt1; RING finger-containing subunit). Whereas Skp1, Cul1, and Rbx1 are common to all SCF complexes, the F-box protein is variable (with ∼70 such proteins having been identified in humans) and confers substrate specificity.Fbxo45 is an F-box protein that was originally isolated as an estrogen-induced protein (47). Human and mouse Fbxo45 genes comprise three exons and possess several consensus binding sequences for the estrogen receptor in the promoter region. Fbxo45 mRNA is rapidly induced on exposure of MCF-7 cells to 17β-estradiol (47). FSN-1, the Caenorhabditis elegans ortholog of Fbxo45, binds to RPM-1 (regulator of presynaptic morphology 1) together with CUL-1 and SKR-1, the C. elegans orthologs of mammalian Cul1 and Skp1, respectively (21, 46). RPM-1 belongs to an evolutionarily conserved family of proteins (the PHR family) that include Highwire (HIW) (Drosophila melanogaster), Esrom (Danio rerio), Phr1 (Mus musculus), and protein associated with Myc (PAM) (Homo sapiens), each of which contains a RING-finger domain that is required for its E3 activity (7, 20, 21, 27, 44). Complete loss of function of fsn-1 in C. elegans results in defects that are characterized by the simultaneous presence of overdeveloped and underdeveloped neuromuscular junctions (NMJs) and which are similar to, but not as pronounced as, those observed in rpm-1^−/− mutants. These genetic findings support the notion that the functions of FSN-1 and RPM-1 are partially overlapping (21).Although PHR family members interact with many potential targets (11, 24, 26, 31), genetic data have shown that one key substrate of RPM-1 and HIW is the mitogen-activated protein kinase kinase kinase known as DLK (dual leucine zipper kinase) in C. elegans and known as Wallenda in D. melanogaster, respectively. The abundance of this kinase is increased in rpm-1 or hiw mutants, and synaptic defects in the mutant worms and flies are suppressed by a loss of DLK or Wallenda. Furthermore, an increase in the level of DLK or Wallenda is sufficient to phenocopy the synaptic defects of the rpm-1 or hiw mutants (5, 27). PAM has also been shown to catalyze the ubiquitylation of tuberin (TSC2) and to regulate signaling by mTOR (mammalian target of rapamycin) in human cells (12).To elucidate the physiological functions of Fbxo45 in mammals, we have now generated mice deficient in this protein. Analysis of the mutant mice revealed that Fbxo45 is required for normal neuromuscular synaptogenesis, axon pathfinding, and neuronal migration. Moreover, we found that Fbxo45 does not form an authentic SCF complex as a result of an amino acid substitution in the F-box domain, and we identified PAM as a binding partner of Fbxo45. The phenotype of Fbxo45^−/− mice was found to be similar to that of Phr1^−/− mice, especially with regard to the defects of neuromuscular synapse formation and of axon navigation. Our results indicate that three fundamental processes of neural development— axonal projection, synapse formation, and neuronal migration—may be linked by a common machinery consisting of the Fbxo45-Phr1 complex.     

Human aquaporin adipose (AQPap) gene. Genomic structure, promoter analysis and functional mutation.

Aquaporin adipose (AQPap), which we identified from human adipose tissue, is a glycerol channel in adipocyte [Kishida et al. (2000) J. Biol. Chem. 275, 20896-20902]. In the current study, we determined the genomic structure of the human AQPap gene, and identified three AQPap-like genes that resembled (approximately 95%) AQPap, with little expression in human tissues. The AQPap promoter contained a putative peroxisome proliferator response element (PPRE) at -46 to -62, and a putative insulin response element (IRE) at -542/-536. Deletion of the PPRE abolished the pioglitazone-mediated induction of AQPap promoter activity in 3T3-L1 adipocytes. Deletion and single base pair substitution analysis of the IRE abolished the insulin-mediated suppression of the human AQPap gene. Analysis of AQPap sequence in human subjects revealed three missense mutations (R12C, V59L and G264V), and two silent mutations (A103A and G250G). The cRNA injection of the missense mutants into Xenopus oocytes revealed the absence of the activity to transport glycerol and water in the AQPap-G264V protein. In the subject homozygous for AQPap-G264V, exercise-induced increase in plasma glycerol was not observed in spite of the increased plasma noradrenaline. We suggest that AQPap is responsible for the increase of plasma glycerol during exercise in humans.